CN101283101A - Method for culturing microorganisms - Google Patents

Method for culturing microorganisms Download PDF

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CN101283101A
CN101283101A CNA2006800370178A CN200680037017A CN101283101A CN 101283101 A CN101283101 A CN 101283101A CN A2006800370178 A CNA2006800370178 A CN A2006800370178A CN 200680037017 A CN200680037017 A CN 200680037017A CN 101283101 A CN101283101 A CN 101283101A
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ethanol
microorganism
aforementioned
substratum
gene
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A·艾金森
R·克里普斯
K·埃利
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TMO BIOTEC Ltd
TMO Renewables Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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Abstract

The present invention is a method for the production of thermophilic microorganisms suitable for the production of ethanol, comprising: (i) culturing a thermophilic microorganism under aerobic or anaerobic conditions in a suitable culture media; and (ii) incorporating amounts of ethanol into the culture media to induce ethanol tolerance.

Description

The method of culturing micro-organisms
Technical field
The present invention relates to produce the alcoholic acid microorganism that is suitable for producing as the fermentation using bacteria product.
Background technology
According to bacterial species and envrionment conditions, bacterial metabolism can take place by various different mechanisms.Heterotrophic bacterium (comprising all pathogenic agent) obtains energy by oxidation of organic compounds, and carbohydrate (particularly glucose), lipid and protein are modal oxidized compounds.The bio-oxidation of utilizing bacterium that these organic compound are carried out finally can be synthesized the ATP as the chemical energy source.This process also can generate bacterial cell and carry out the required simple more organic compound (precursor molecule) of biosynthesizing reaction.The general process that bacterial metabolism is fit to substrate is glycolysis-, glycolysis-be a series of be the reaction that pyruvic acid generates ATP simultaneously with conversion of glucose.The whereabouts of pyruvic acid can change according to microorganism and envrionment conditions in the process of generation metabolizable energy.Main pyruvic acid reaction has three kinds.
The first, under aerobic conditions, many microorganisms can utilize tricarboxylic acid cycle and pyruvic acid is converted into acetyl-CoA and come generate energy under the catalysis of pyruvic oxidase (PDH).
The second, under anaerobic, some producing and ethanol biology can be by becoming acetaldehyde with the pyruvic acid decarboxylation under the catalysis of pyruvic carboxylase (PDC), and utilize NADH that acetaldehyde reduction is carried out ethanol fermentation for ethanol subsequently under the catalysis of alcoholdehydrogenase (ADH).
The 3rd process is by serum lactic dehydrogenase (LDH) catalysis, and pyruvic acid is converted into lactic acid.
Next life, producing and ethanol was very interested in using microorganism---to use natural microorganism or the use of carrying out anaerobically fermenting to introduce the recombinant microorganism of related gene in the alcohol production---for people.Although obtained certain success aspect these microorganisms producing ethanol utilizing, the increase of usually ethanol concentration can make fermentation impaired, particularly when the alcohol resistance level of microorganism is hanged down.
The someone proposes to use thermophilic bacterium to carry out alcohol production, uses the advantage of thermophilic bacterium to be and can to ferment under comparatively high temps, and this makes and can be under the temperature more than 50 ℃ the ethanol that the produced form with steam be shifted out; This also makes and can utilize high glucose concentration to ferment.Yet can problem be find can effectively produce the suitable thermophilic bacterium of alcoholic acid.
WO01/49865 discloses a kind of gram positive bacterium that has been transformed the heterologous gene of coding pyruvic carboxylase and had a natural alcoholdehydrogenase function and has been used for alcohol production.Described bacterium is the bacillus (Bacillus) of thermophilic, can make the lactate dehydrogenase gene inactivation come this bacterium is modified by inserting transposon.Disclosed bacterium all derives from Bacillaceae bacterial strain LLD-R among the WO01/49865, and it is a kind of sporulation defective bacterial strain that spontaneously grows from culture, and in this bacterial strain the ldh gene by spontaneous mutation or chemomorphosis inactivation.Disclosed bacterial strain LN and TN are the derivative strains of the improvement of LLD-R bacterial strain.Yet whole bacterial strains all contains the restricted system of Hae III type, and described restricted system hinders plasmid to transform the conversion that also therefore stops in the non-methylate DNA.
WO01/85966 discloses by methylating in the body to overcome the microorganism that restricted problem prepares.This need be transformed into Hae III methyltransgerase LLD-R, LN and the TN bacterial strain from bacterium aegyptiacum (Haemophilusaegyptius).Yet LLD-R, LN and TN bacterial strain are unsettled mutant and can spontaneously reply wild type strain into lactic acid producing, particularly in the environment of low pH and high glucose concentration.This can cause tunning to become lactic acid from ethanol, makes described bacterial strain not be suitable for alcohol production.
WO02/29030 discloses the LLD-R bacterial strain and derivative strain comprises a naturally occurring insertion element (IE) at the ldh gene coding region.It all is unsettled this insertion element being changed over to (with producing) swivel base of ldh gene and gene inactivation subsequently, thereby causes replying.The solution that this is provided is that plasmid DNA is incorporated in the described IE sequence.
Therefore, in the art, the generation that is used for the microorganism of the alcohol production bacillus spp. microorganism that depends on the chemomorphosis that the laboratory is produced is modified, is handled these microorganisms and further modify described microorganism so that plasmid DNA is incorporated into described IE sequence with methylation method in the body.This method is complicated and uncertain, and also has handling problem how to use described bacterial strain.
So the microorganism that is used for alcohol production that need improve.
Summary of the invention
According to a first aspect of the invention, a kind of method that is used to produce the thermophilic microorganism that is suitable for alcohol production comprises:
(i) under aerobic or the anaerobic condition, in the substratum that is fit to, cultivate thermophilic microorganism; With
(ii) the ethanol that consumption is increased gradually mixes described substratum to induce alcohol resistance.
Embodiment
The present invention is based on and handles thermophilic microorganism so that described microorganism tolerates more to ethanol, and therefore can produce ethanol better.The alcohol resistance that increases described microorganism makes described microorganism to tolerate more the ethanol that is produced in its fermenting process.This makes and can improve fermentation.
The described method that is used for thermophilic microorganism production is included under aerobic or the anaerobic condition, cultivates thermophilic microorganism and adequate amount of ethanol is mixed described substratum to induce alcohol resistance at the substratum that is fit to.In one embodiment, increasing ethanol in described substratum is to carry out in the mode of increment in time and usually, so that the ethanol that described microorganism increases in can acclimatizing culture medium.Preferably ethanol is incorporated into the final concentration that is up to 3%w/v, then alcohol concn is increased 0.5%w/v or still less the alcohol concn in substratum be at least 6%w/v, more preferably at least 7.5%.In one embodiment, initial substratum contains the ethanol of 3%w/v, then its increment with 0.5% is increased to 6%w/v, is increased to 7.5%w/v with 0.25% increment then.
In this step, can monitor cell density and continue growth to guarantee cell.Preferably, if cell density (measuring by OD600nm) descends above 25% and with alcohol concn increase continuing decline, then alcohol concn is reduced to a last maximum concentration, and is continuing with recovery media before the Ethanol Treatment.
The method that a kind of alternative being used for has the thermophilic microorganism generation of higher ethanol tolerance is included under aerobic or the anaerobic condition, cultivates described thermophilic microorganism at the substratum that is fit to.In case culture reaches stable state---at this moment, the growth of described biology has reached constant rate of speed (determining by OD600nm)---then in described substratum, once add adequate amount of ethanol so that ethanol reaches a specific desired concn, 10% or 20%w/v of for example described substratum setting working volume.With low dilution rate (preferred 0.08-0.15h -1) continue this cultured continuously, make alcohol concn slowly reduce and recovered (measuring) by OD600nm until described thermophilic initial growth speed.At this moment, in described substratum, once add the ethanol of an amount of (identical) again, so that ethanol reaches a specific desired concn with primary add-on.Repeat the described process that makes described culture recover initial growth speed, and further add ethanol until finding that described culture recovers rapidly in batches, this process cost was less than 24 hours.One of two kinds of results can appear in this moment.Cultivate screening ethanol tolerance bacterial strain from this culture by under required alcohol concn (preferably being higher than 7.5%w/v), going down to posterity, perhaps join more substantial ethanol in the described culture and repeat to add the process that growth velocity is recovered after the ethanol.
Under 55 ℃-65 ℃, described microorganism is grown in containing the defined medium of limitting carbon substrate, pH is 6.0-7.5 (preferred 6.3-7.2), dilution rate is 0.08-0.5.
In batch culture, can implement a kind of method that is similar to method mentioned above, growth and add ethanol in early days in containing any suitable substratum of excess carbon at logarithmic phase.Use the cell in initial incubation thing logarithmic phase latter stage to inoculate new flask then, and add the ethanol of increment once more in logarithmic phase in early days.Repeat this step, repeat in described substratum, to add in early days the ethanol of increment simultaneously in logarithmic phase.
Can modify with related expression of gene in interruption or the enhancing bio-chemical pathway relevant thermophilic microorganism used among the present invention, for example interrupt lactate dehydrogenase gene with the ethanol biosynthesizing.The direction that this can guide the pyruvic acid metabolism to deviate to produce lactic acid and towards producing the alcoholic acid direction, and in the negative mutant of lactic acid, observe the ethanol level and raise.
Make the lactate dehydrogenase gene inactivation help to prevent that pyruvic acid is degraded into lactic acid, and therefore utilize pyruvic carboxylase and alcoholdehydrogenase to promote (under the condition of being fit to) pyruvic acid to be degraded into ethanol.
Described wild-type microorganisms can be any thermophilic microorganism, but described microorganism is preferably Bacillaceae kind (Bacillus spp).Described microorganism particularly preferably belongs to kind of (a Geobacillus species), particularly hot Polyglucosidase ground bacillus (Geobacillusthermoglucosidasius) for ground bacillus.
Described microorganism can be " wild-type ", and promptly they are not the mutant that the laboratory produces.Described microorganism separates from estimating to contain thermophilic environmental sample.Isolating wild-type microorganisms has the alcoholic acid of production ability, but because not modified, main tunning may be a lactic acid.Also at high temperature utilize the ability of hexose and/or pentose growth to come they are screened according to strain isolated.Also can use non-wild-type mutant microorganism.
Preferably, microorganism of the present invention has some and can make described microorganism be used to the required feature of fermenting process.Described microorganism should preferably not have restricted system, has therefore avoided methylated needs in the body.In addition, described microorganism should have C5 and C6 sugar (comprising cellobiose and the starch) ability as substrate of utilizing.Preferably, described microorganism can transform with high frequency.In addition, the growth rate of described microorganism in cultured continuously should be at 0.3 hour -1More than.
Described microorganism can be thermophile and can grow in 40 ℃-85 ℃ temperature range.Preferably, described microorganism grows in 50 ℃-70 ℃ temperature range.In addition, described microorganism grows under pH 6.5 or lower condition, and particularly growth is an ideal under the condition of pH 6.5-pH 4.5.
The nucleotide sequence of serum lactic dehydrogenase is known now.Use this sequence, those skilled in the art's energy target lactate dehydrogenase gene is to realize the inactivation of this gene by different mechanisms.Preferably, lactate dehydrogenase gene is by inserting that transposon comes inactivation or preferably coming inactivation by a part that lacks this gene order or this gene order.Preferably by disappearance, because disappearance has been avoided the difficult problem of gene order reactivate, described this difficult problem of gene order reactivate can often run into when adopting the transposon inactivation method.In a preferred embodiment, described lactate dehydrogenase gene comes inactivation by integrating a temperature sensitive plasmid (plasmid pUB190-ldh), and this has realized natural homologous recombination or integration between plasmid and the microbial staining body.According to chromosomal intasome the resistance of antiseptic-germicide (kantlex) is come it is screened.Integration in the lactate dehydrogenase gene can realize by the single exchange recombination event or by twice (or repeatedly) exchange recombination event.
In a preferred embodiment, described microorganism contains allogenic alcohol dehydrogenase gene and allogenic Pyruvate Decarboxylase Gene.These expression of heterologous genes can cause the generation of some enzymes like this, and described enzyme can redirect described metabolism so that ethanol becomes main tunning.These genes comprise Zymomonas (zymomonas) kind available from the microorganism that carries out aerobic fermentation usually, comprise motion fermentation sporangium (zymomonas mobilis).
Be used for preparing and be known, Ingram et al for example, Biotech ﹠amp the method that these genes are introduced microorganisms; BioEng, 1998; 58 (2+3): 204-214 and US 5916787, its content all by reference mode is included this specification sheets in.It will be understood by those skilled in the art that described gene can or be integrated in the karyomit(e) with the plasmid introducing.
According to selected thermophilic microorganism, can under the culture condition of routine, cultivate microorganism of the present invention.Require to select substrate, temperature, pH and other growth conditions according to known cultivation, for example referring to WO01/49865 and WO01/85966.The cultivation and the fermentation condition that are fit to have been shown in the table 1,2 and 3:
Table 1
Chemical reagent Volume/L Final concentration
NaH 2PO 4·2H 2O 25mM
K 2SO 4 10mM
Citric acid H 2O 2mM
MgSO 4·7H 2O 1.25mM
CaCl 2·2H 2O 0.02mM
The vitriol trace element solution 5ml See the following form
Na 2MoO 4·2H 2O 1.65μM
Yeast extract 10g
Defoamer 0.5ml
Additive behind the autoclaving
4M urea 25ml 100mM
1% vitamin H 300μl 12μM
20% glucose 2 50ml 1％
Table 2
Vitriol trace element liquid storage
Chemical reagent gl -1(ml) gl -1(ml) Substratum concentration
The vitriol oil 5ml 50ml
ZnSO 4·7H 2O 1.44 14.4 25μM
FeSO 4·7H 2O 5.56 55.6 100μM
MnSO 4·H 2O 1.69 16.9 50μM
CuSO 4·5H 2O 0.25 2.5 5μM
CoSO 4·7H 2O 0.562 5.62 10μM
NiSO 4·6H 2O 0.886 8.86 16.85μM
H 3BO 3 0.08
Deionized water (end) 1000ml 10 liters
Table 3
The fermentor tank condition
Inoculum 10％v/v
Volume 1000ml
Temperature 60℃
PH (7.0 regulating) with NaOH
Inflation 0.4vvm
N 2Stream 0.05lpm
Stir 400rpm
Substratum The urea sulfate CDM that is used for fermentor tank
Sugar is supplied with The glucose of 100ml 50%
Defoamer
The present invention will be described by following embodiment with reference to accompanying drawing.
Embodiment
Prepared following substratum:
SAM2-perL
Yeast extract 1.0g
Tryptones 0.5g
NH 4Cl 1.0g
NaH 2PO 4 0.5g
MgSO 4·7H 2O 0.2g
KCl 0.2g
MnCl 24H 2O 3mg (adds the storage of 100 μ L 30mg/mL
Liquid)
CaCl 22H 2O 5mg (adds the storage of 100 μ L 50mg/mL
Liquid)
PIPES damping fluid 12.096g
Be dissolved in the cumulative volume behind the distilled water: 950mL, with NaOH or H 2SO 4PH is transferred to 7.0.
Autoclaving.
After the cooling, add 2.5mL vitriol trace element liquid storage (referring to table 2), and the sugar soln of 50mL20%w/v filtration sterilization.
US (urea salt) substratum (USM) of improvement
Glucose 10.0g/L
Yeast extract 0.8g/L
Citric acid 0.42g/L
MgSO 4 0.31g/L
NaH 2PO 4 3.1g/L
K 2SO 4 3.5g/L
Urea 3.0g/L
CaCl 2 2mg/L
Na 2MoO 4 4mg/L
Trace element solution (table 2) 5.0ml/L
For solid medium, add the 20.0g/L Bacto-agar.
Before sterilization, proofread and correct to pH 7 with 3M NaOH.
The TGP substratum
Microbial culture Tryptones 17.0g/L
Soy peptone 3.0g/L
NaCl 5.0g/L
K 2HPO 4 2.5g/L
Sodium.alpha.-ketopropionate 4.0/L
Glycerine 4.0mL/L
For solid medium, add the 20.0g/L Bacto-agar.Before sterilization, substratum is proofreaied and correct to pH 7 with 3MNaOH.
The alcohol resistance of having tested a kind of wild-type biology (NCIMB 11955) is to determine starting point.This biological growth is spent the night (LB agar plate, 60 ℃) and use a bacterium colony inoculate overnight culture (100mL USM, 1% glucose, 60 ℃, 250rpm).Use this culture to inoculate then to contain 0,1,2,3 or 4% alcoholic acid and establish three multiple flasks respectively, make its growth 36 hours then, measure afterwards growth (50mL USM, 1% glucose, 60 ℃, 250rpm).The result is shown in Figure 1, and the result shows that NCIMB 11955 can not tolerate and is higher than 4% ethanol.
Utilize this result basis as a comparison, experimentize and increase to the ethanol of 8%v/v with alcohol resistance mutant TM89.
Fermentation process
Microorganism: Gt TM-89
Inoculum: 50ml 2xYT culture (7%v/v)
Equipment: LH glass fermentor tank (700ml working volume)
Use Anglicon Controlling System controlled temperature
And pH
The use magnetic stirring apparatus mixes
Set(ting)value: temperature: 60 ℃
pH：6.80
Air: 0.2-0.4vvm
Stirring velocity: 225rpm
Use Watson Marlow pump (0-100ml/
Hour) the control flow velocity
Substratum: SAM2 2% glucose, 0.05% organic froth breaking
Agent (referring to cultivating base table)
Use 10%NaOH to regulate PH
Amount of alcohol added (spike): 75ml or 150ml ethanol (add
10-20％)
The strategy that adopts is:
1) reaches stable state and measurement product productive rate/sugared utilization ratio; With
2) ethanol is added into culture, makes culture recover (constantly washing ethanol in time off), take out sample and prepare the glycerine liquid storage, as required repeating step (2).
For the assessment alcohol resistance, developed following proposal.
1. 5ml TGP meat soup is joined in two aseptic general test tubes.
2. TM-89 and the TM89-1 glycerine liquid storage with 100 μ l joins respectively in each test tube.
3. 60 ℃ of following shaking culture 5-6 hours (therefore activated cell, this moment, they should be in logarithmic phase).
4. measure the OD of every portion of meat soup 600(hence one can see that initial OD 600).
5. prepare 11 aseptic general test tubes that contain 10ml TGP meat soup, described TGP meat soup contains the ethanol that concentration is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% (v/v) respectively, and each TM-89 bacterial strain is established two repetitions (i.e. 44 test tubes).
6. with 100 each test tube of μ l cell inoculation, described cell is taken from the 5ml TGP meat soup of the correspondence that is in the 1+2 stage.
7. spend the night 60 ℃ of following shaking culture.
8. from each test tube, take out 1ml, by dilution in 1: 5 and with H 2O is blank measure OD 600
Interpretation of result is as follows:
(1) use the optical density(OD) of Jencons spectrophotometer measurement culture [according to A 600X0.3 calculates cell concn (g/L)].
(2) use blood-glucose meter (Roche) measure glucose concentration.
(3) use is measured alcohol concn based on the mensuration test kit (being provided by R-Biopharm) of enzyme.
The result is shown in Figure 2.With compare with the initial TM89 bacterial strain that the TGP that contains a series of concentration ethanol cultivates with TGP, strain separated demonstrates higher OD value always during fermentation ends.Ethanol is the alcohol resistance that the significant difference of 5% o'clock growth has shown improvement.

Claims (12)

1. a production is suitable for the method for the thermophilic microorganism of alcohol production, comprising:
(i) under aerobic or the anaerobic condition, in the substratum that is fit to, cultivate thermophilic microorganism; With
(ii) adequate amount of ethanol is mixed described substratum to induce alcohol resistance.
2. the process of claim 1 wherein that in described substratum increment ground adds ethanol gradually, and before back increment ground adding ethanol, make described microorganism adapt to added ethanol.
3. claim 1 or 2 method wherein are incorporated into final concentration with ethanol and are at least 3%w/v.
4. the method for aforementioned each claim wherein is incorporated into final concentration with ethanol and is at least 6%w/v.
5. the method for aforementioned each claim wherein is incorporated into final concentration with ethanol and is at least 7.5%w/v.
6. the method for aforementioned each claim, wherein ethanol is to mix with 0.5%w/v or increment still less.
7. the method for aforementioned each claim, wherein ethanol is incorporated in the described substratum in early days in logarithmic phase.
8. the method for aforementioned each claim, wherein said microorganism has the lactate dehydrogenase gene of inactivation.
9. the method for aforementioned each claim, wherein said microorganism does not contain restricted system.
10. the method for aforementioned each claim, wherein said microorganism is hot Polyglucosidase ground bacillus (Geobacillus thermoglucosidasius).
11. the method for aforementioned each claim, wherein said microorganism contain non-natural pdc gene.
12. the method for aforementioned each claim, wherein said microorganism contain non-natural adh gene.
CNA2006800370178A 2005-10-06 2006-10-05 Method for culturing microorganisms Pending CN101283101A (en)

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BRPI0717228A2 (en) * 2006-09-28 2013-10-08 Tmo Renewables Ltd THERMOPHILIC MICROORGANISMS FOR ETHANOL PRODUCTION
CN101848924A (en) 2007-05-09 2010-09-29 马斯科马公司 Gene knockout mesophilic and thermophilic organisms, and methods of use thereof
GB0715751D0 (en) 2007-08-13 2007-09-19 Tmo Renewables Ltd Thermophilic micro-organisms for ethanol production
GB0820262D0 (en) 2008-11-05 2008-12-10 Tmo Renewables Ltd Microorganisms
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